The invention relates to the preparation of porcine cardiac valve leaflets for implantation into patients.
Diseased cardiac valves can be replaced with either mechanical or biological tissue prostheses. Mechanical valves are more durable but require long-term anti-coagulation therapy with coumadin to prevent surface clotting. Risks associated with anticoagulation therapy, such as bleeding or thromboembolism, are of concern to many physicians and their patients. Biological tissue valves as protheses, on the other hand, do not require anticoagulation therapy.
Xenografts are a desirable source of biological tissue, but xenotransplantation is currently performed using highly preserved tissues to avoid rejection. The presentation techniques eliminate tissue viability and may result in post-implant calcification that shortens the half-life of the implant. Therefore, the usefulness of preserved implants is limited by durability secondary to structural degeneration and/or calcification.
The first reported use of formaldehyde on valves was to denature and stiffen elastic fibers in human homografts (Paneth et al, Thorax, 21:115-7. 1966). Initial formaldehyde preservation of porcine valves was performed by O""Brien, who used the solution in 1967 for xe2x80x9cboth preservation and sterilizationxe2x80x9d (O""Brien, J. Thoracic Cardiovasc. Surgery, 53:392-7. 1967). Early investigators hypothesized that the fixative denatured the proteins of the graft, thereby minimizing rejection. (See: Hamilton et al, Thorax, 23:239-48. 1968; Paneth et al, Thorax, 21:115-7. 1966). Currently, biological tissue valves are constructed with glutaraldehyde-fixed porcine or bovine tissues, which may explain the considerably reduced biological half-life of these xenotransplanted valves as compared to native human valves.
The transplantation of porcine hearts into humans prompts a violent hyperacute rejection similar to that seen in ABO-incompatible transplantation, in which the graft becomes cyanotic, and edematous minutes after implantation (Chen et al, In: Simon D, Roberts C, eds. Vascular Disease and Injury: Preclinical Research. Ottowa: Humana, in press, 2000). The extensive microvascular thrombosis of hyperacute rejection is the result of IgM-triggered, complement Membrane Attack Complex (MAC)-mediated graft destruction. This mechanism of pig-to-human xenotransplantation rejection was not understood until 1987, when it was shown that humans constituitively synthesize natural IgM antibodies against a porcine molecule, galactose xcex1-1,3 galactose (Galili et al, Proc Natl Acad Sci USA, 84:1369-73. 1987).
In contrast, is the novel and unexpected finding that isolated porcine cardiac valve leaflets lack galactose-xcex1 1,3-galactose expression and are therefore non-immunogenic in humans and other primates. This surprising finding is the basis for preparing porcine cardiac valve leaflets with little or no contact with fixative agents. These unfixed and minimally fixed tissues can be successfully used as xenografts. According to the invention, because these isolated porcine cardiac valve leaflet tissues show no immunogenicity in the host, they can be used either untreated or minimally treated with fixatives, thereby increasing implant viability and durability over that of past implants, and overcoming some of the limitations of xenotransplantation.
The invention is based on the surprising finding that porcine cardiac valve leaflets do not express galactose-xcex1 1,3-galactose and are not immunogenic when implanted into primates. Accordingly, the invention is related to preparations of isolated porcine cardiac valve leaflets for implantation into patients. The invention also relates to methods for preserving isolated porcine cardiac valve leaflets prior to implantation into patients, such as fixation-free, minimal fixed, and/or cryopreserved. In addition, the invention relates to methods of treating a patient by implantation into the patient of a fixation-free, minimal-fixed and/or cryopreserved porcine cardiac valve leaflet or leaflets.
In one aspect of the invention a medical preparation is provided. The preparation contains an isolated porcine cardiac valve leaflet, free of contact with an exogenous fixative. In one embodiment the preparation is packaged sterile in a container in an extracellular solution. In another embodiment, the extracellular solution contains a cryoprotectant agent. In another embodiment, the preparation is frozen.
Another aspect of the invention provides a kit for implanting a cardiac valve leaflet into a patient. The kit is a container including an isolated porcine cardiac valve leaflet, free of contact with a fixative, packaged sterile in a container in an extracellular solution. The kit also contains written instructions for processing the isolated porcine cardiac valve leaflet for implantation into a patient. In one embodiment of the kit, the extracellular solution contains a cryoprotectant. In other embodiments, the leaflet is frozen.
Yet another aspect of the invention provides a medical preparation. The preparation is an isolated porcine cardiac valve leaflet, contacted for a time with an amount of an exogenous fixative effective to prolong shelf-life versus an uncontacted porcine cardiac valve leaflet, but wherein said time and amount are insufficient to induce a calcification level of a percentage which is more than 80% of an identical-interval, post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde. The preparation can be packaged sterile in a container in an extracellular solution. A preferred percentage of calcification is less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 2%, or 1%, of the level of calcification characteristic of an identical-interval, post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde and implanted. In one embodiment, the contact with the fixative is for equal to or less than 120, 60, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5 minutes. In one embodiment, the fixative is gluteraldehyde. In another embodiment, the fixative is gluteraldehyde and contacts the preparation for 2 hours or less. In another embodiment, the fixative is 0.60%, 0.55%, 0.45%, 0.35%, 0.25%, 0.15%, 0.10%, 0.05%, 0.01% or 0.001% gluteraldehyde. In another embodiment, the fixative is ethanol. In another embodiment, the fixative is ethanol and contacts the preparation for 1 hour or less. In yet another embodiment, the fixative is acetone. In another embodiment, the fixative is acetone and contacts the preparation for 1 hour or less. In another embodiment, the extracellular solution contains a cryoprotectant agent. In another embodiment, the preparation is frozen.
Another aspect of the invention provides a cryopreserved implant that is a frozen isolated porcine cardiac valve leaflet with a predicted shelf-life of 13 months or more and a predicted post-implantation half life of 13 years or more when thawed and implanted into a patient. In one embodiment, the predicted post-implantation half life of the implant is determined by the level of leaflet calcification post implantation. In another embodiment, the predicted level of implanted valve leaflet calcification level is less than 80% of the identical-interval post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde. The leaflet can be packaged sterile in a container. The range of calcification level, the range of times of exposure, and preferred fixatives and fixative solutions are as described above herein.
Another aspect of the invention provides a kit for implanting a cardiac valve leaflet into a patient. The kit is a container including an isolated porcine cardiac valve leaflet, contacted for a time with an amount of an exogenous fixative effective to prolong shelf-life versus an uncontacted porcine cardiac valve leaflet, but wherein said time and amount are insufficient to induce a percentage of calcification level of 80% of the identical-interval, post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde; packaged sterile in a container in an extracellular solution. A preferred range of calcification level, the range of times of exposure to fixative, and preferred fixatives and fixative solutions are as described above herein. The kit also contains written instructions for processing the isolated porcine cardiac valve leaflet for implantation into a patient. In one embodiment, the extracellular solution contains a cryoprotectant agent. In another embodiment, the preparation is frozen.
Another aspect of the invention is a method of preparing an isolated porcine cardiac valve leaflet for implantation into a patient. In the method, an isolated porcine cardiac valve leaflet is contacted for a time with an amount of an exogenous fixative effective to prolong shelf-life versus an uncontacted porcine cardiac valve leaflet, but wherein said time and amount are insufficient to induce a percentage of calcification level of 80% of the identical-interval, post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde, and the contacted isolated leaflet is packaged sterile in a container in an extracellular solution. A preferred range of calcification level, range of times of exposure to fixative, and preferred fixatives and fixative solutions are as described above herein. In one embodiment, the extracellular solution contains a cryoprotectant agent. In yet another embodiment, the isolated porcine cardiac valve leaflet is frozen.
Another aspect of the invention is a method of preparing an isolated porcine cardiac valve leaflet for implantation in a patient. In the method, the isolated porcine cardiac valve leaflet is processed for implantation without contact with an exogenous fixative. The leaflet can be held or packaged fresh, sterile in a container in an extracellular solution. In another embodiment, the extracellular solution contains a cryoprotectant agent. In yet another embodiment, the isolated porcine cardiac valve leaflet is frozen.
Another aspect of the invention is a method of treating a patient that is implanting into the patient one or more isolated porcine cardiac valve leaflets which have been contacted for a time with an amount of an exogenous fixative, wherein said time and amount are insufficient to induce a percentage of calcification level of 80% of the identical-interval, post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde. A preferred range of calcification level, range of times of exposure to fixative, and preferred fixatives and fixative solutions are as described above herein. In one embodiment, the leaflet has not previously been frozen. The leaflet may be obtained fresh, i.e., recently harvested without overnight storage. In another embodiment, the method involves thawing a leaflet, the leaflet having been stored frozen.
Another aspect of the invention is a method of treating a patient that is thawing and implanting into the patient one or more isolated porcine cardiac valve leaflets which have been contacted for a time with an amount of an exogenous fixative, wherein said time and amount are insufficient to induce a percentage of calcification level of less than 80% of the identical-interval, post-implantation calcification level of a porcine cardiac valve leaflet contacted 6 hours with 0.65% gluteraldehyde. A preferred range of calcification level, range of times of exposure to fixative, and preferred fixatives and fixative solutions are as described above herein.
Another aspect of the invention is a method of treating a patient that is implanting one or more isolated porcine cardiac valve leaflets which have been prepared for implantation without contact with an exogenous fixative. The method can involve obtaining a leaflet that may be fresh, i.e., recently harvested without overnight storage. The leaflet also may have been stored in a non fixative storage solution. In one embodiment, the leaflet is stored at bout 4xc2x0 centigrade. In another embodiment, the method involves thawing the leaflet, the leaflet having been stored frozen.
For any of the forgoing embodiments, an exogenous fixative includes any tissue fixative agent or means, known to those of ordinary skill in the art. Thus, an exogenous fixative preferably is one selected from the group consisting of chemical or irradiation fixatives.